PROJECT SUMMARY Intercellular communication mediated by Cx46 and Cx50 gap junction channels is central to the transport of ions into the lens and to the maintenance of lens transparency. Studies on KO mice and on Cx46 and Cx50 mutations have established that the absence of gap junctional coupling leads to cataracts. The main focus of this proposal is to examine the role of gap junction (GJ) channels in the development of age-related nuclear cataracts. A key factor that influences nuclear cataract formation is the abrupt age-dependent decline in the levels of reduced glutathione (GSH) in the lens nucleus. This reduction in GSH levels is not observed in the outer cortex and therefore has been attributed to the formation of a barrier to the diffusion of GSH that develops with age. We have used electrophysiological techniques to show that that Cx46 and Cx50 channels are permeable to GSH. Additional studies indicated that GSH is likely to diffuse via Cx46 GJs from the cells in the outer cortex, where the anti-oxidant is synthesized, to the metabolically quiescent cells in the lens nucleus. In this proposal, we will examine the role of GJ channels in: (1) the establishment of a reducing environment in the lens nucleus, and (2) mediating the shift to an oxidized environment that occurs with aging. We will use imaging and mass spectrometry methods to directly measure the variation in the glutathione redox potential from the lens periphery to the center in the intact lens. In Aim 1, we will assess the influence of coupling levels on the spatial profile of glutathione redox potential in the intact lens. In Aim 2, we will determine the permeability of gap junctions to other metabolites known to be essential for the overall redox state of cells deep in the lens. In Aim 3, we will examine the relationship between GJ channel functionality and changes in the glutathione redox potential that occur with age. Specifically, we will determine whether post-translational modifications to Cx46 and Cx50 accumulate with age, leading to a reduction in GJ coupling, and ultimately to the development of the barrier to GSH diffusion. These studies thus make a direct contribution to the understanding of the mechanisms underlying the formation of ARN cataracts, and will serve to further highlight the key role of GJ channels in maintenance of lens transparency.